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To identify the genes for biosynthesis of the off-flavor terpenoid alcohol, 2-methylisoborneol (2-MIB), the key genes encoding monoterpene cyclase were located in bacterial genome databases by using a combination of hidden Markov models, proteinCfamily search, and the sequence alignment of their gene products. A3(2) (21) and (22). Another sesquiterpene cyclase generating epi-isozizaene that could be an intermediate of albaflavenone biosynthesis, epi-isozizaene synthase (SCO5222p) of A3(2), has been characterized (23). Fig. 1. Structures of microbial volatile terpenoid metabolites, 2-MIB (and genome data, draft sequence data, and limited sequence data from a linear plasmid pKSL of [putative terpene cyclase and methyltransferase genes were found near the genes for modular polyketide synthases (30)] by searching on the basis of the profile hidden Markov models using a model of PF03936 913822-46-5 manufacture (terpene synthase family, metal binding domain). The E-value used was <10?5 for setting the parameter of hidden Markov models search because proteins selected using >10?5 E-value were also selected by other Pfam models with lower E-values. Of 1 1,922,990 proteins, 41 proteins were selected with E-value ranges from 2.2 10?6 to 1 1.8 10?82. These proteins were classified into three major groups on the basis of phylogenetic analysis (Fig. 2). The sesquiterpene cyclases, pentalenene, germacradienol/geosmin and epi-isozizaene synthases, were classified in group II. Group III contained diterpene cyclase in terpentecin biosynthesis of (31) and undefined terpene cyclase of sp. PCC 7120; Ava_1982 (322 aa; … As shown in Fig. 3, two conserved motifs of the proteins in group I were located near the C terminus, in comparison with those of sesquiterpene and diterpene cyclases. The distance between the first and second conserved motifs of the Rabbit Polyclonal to CDK5 proteins in group I (112 aa) was longer than those of sesquiterpene and diterpene cyclases (104C106 aa). The first motif in sesquiterpene cyclases was acid-rich domain with 913822-46-5 manufacture a high proportion of aromatic amino acids, CFFxxDDxxDC (pentalenene and epi-isozizaene synthases) or CFxFDDHFLEC (germacradienol/geosmin synthase). Although the first motif in diterpene cyclase (CLIVNDDRWDC) and the proteins of group I (CxVDDxxx[DE]C) also possessed an acid-rich domain, the content of aromatic amino acids was lower than that in sesquiterpne cyclases. The second motif in all proteins were conserved, CxxNxxxSxxxEC, in which the triad of residues in bold has also been implicated in the binding of magnesium ion (33, 34, 36). Because the SAV (deduced amino acid sequence of truncated terpene cyclase; 1,245,680 to 1 1,246,412 nt of the genome) was defined as a pseudogene of A3(2), (SCAB5041), formed an operon with a gene encoding SAM-dependent methyltransferase (Fig. 913822-46-5 manufacture 4). Furthermore, genes encoding cyclic nucleotide-binding protein were also located upstream of these seven monoterpene cyclase genes. On the other hand, methyltransferase gene(s) was/were not found in or around monoterpene cyclase genes predicted in and (SCAB82161). These two monoterpene cyclases would be involved in other monoterpenoid metabolite biosynthesis. Interestingly, a similar methyltransferase gene, SAV983, was found in genome data (http://avermitilis.ls.kitasato-u.ac.jp/). Furthermore, truncated cyclic nucleotide-binding protein and monoterpene cyclase genes were found upstream of SAV983. The truncation will be induced by a deletion mutation, and deduced polypeptide of truncated gene product lacked the second conserved metal-binding motif (Fig. 3 would be capable of producing 2-MIB, but the deletion of the region encoding the second conserved metal-binding motif in monoterpene cyclase gene prevented production of 2-MIB in this organism. Fig. 4. Organization of genes encoding predicted monoterpene cyclases and flanking genes. All predicted monoterpene cyclase genes are located in the chromosomes, but the gene of resided in a giant linear plasmid pKSL. The grayed, filled, and oblique-lined … Production of Volatile Terpenoid Metabolites in Actinomycetes Carrying Predicted Monoterpene Cyclase/Methyltransferase Genes. The results of bioinformatics indicate that seven actinomycetes possess the ability to produce the monoterpenoid metabolite, 2-MIB. Because a grow pathogen 87.22 was not stored in our culture stock, six actinomycete strains were examined. All six strains produced terpenoid metabolites, including sesquiterpenoid alcohol, geosmin, in varying quantity (Fig. 5). Five strains, A3(2), 168) to those of the authentic 2-MIB. Interestingly, did not produce 2-MIB, but homomonoterpene hydrocarbon ([M+], 150) was accumulated as a major component. The hydrocarbon was identical to 2-methylenebornane (2-MB), a dehydration product of 2-MIB in comparison with retention time and mass spectrum of the synthetic sample. Then inspection of mass spectra obtained from the extracts of all strains and allowed us to detect as a minor component. Thus, the monoterpene cyclase of is slightly.